Transglutaminases (EC 2.3.2.13) are enzymes capable of catalyzing an acyl transfer reaction in which a .gamma.-carboxy-amide group of a peptide bound glutamine residue is the acyl donor. Primary amino groups in a variety of compounds may function as acyl acceptors with the subsequent formation of monosubstituted .gamma.-amides of peptide bound glutamic acid. When the .epsilon.-amino group of a lysine residue in a peptide chain serves as the acyl acceptor, the transglutaminases form intramolecular or intermolecular .gamma.-glutamyl-.epsilon.-lysyl crosslinks.
This peptide crosslinking activity has shown useful for a variety of industrial purposes, including gelling of proteins, improvement of baking quality of flour, producing paste type food materia from protein, fat and water, preparation of cheese from milk concentrate, binding of chopped meat product, improvement of taste and texture of food proteins, casein finishing in leather processing etc.
A wide array of transglutaminases have been identified and characterized from a number of animals and a few plant species. The most widely used animal derived transglutaminase, FXIIIa, is a Ca.sup.2+ -dependent multi-subunit enzyme which is product inhibited, properties which are a disadvantage for many industrial applications and for production. A Ca.sup.2+ -dependent transglutaminase from the slime mould Physarum polycephalum has been described in Klein et al., (1992).
Only few microbial transglutaminases have been disclosed, namely tranglutaminases from the species Streptoverticillium mobaraense, Streptoverticillium cinnamoneum, and Streptoverticillium griseocarneum (in U.S. Pat. No. 5,156,956) and from the species contemplated to be Streptomyces lavendulae (in U.S. Pat. No. 5,252,469).
U.S. Pat. No. 5,156,956 discloses that, after an extensive search for transglutaminases including screening a wide range of organisms and more than 5000 isolates of microbial origin, only the above-mentioned three Streptoverticillium species were found to produce transglutaminase. Members of this former genus Streptoverticillium are now generally included within the genus Streptomyces (Kaempfer et al. (1991), and Ochi et al. (1994)).
U.S. Pat. No. 5,252,469 discloses transglutaminase from what was believed to be two related species: Streptomyces sp., and Streptomyces lavendulae. However, from the disclosed data for the contemplated S. lavendulae strain it is evident to the skilled person that the disclosed strain is not S. lavendulae.
Streptoverticillia are classified together in Cluster group F (clusters 55 to 67) of Streptomyces and related genera (Williams et al.). Therefore the known microbial transglutaminases all originate from members of this Cluster group as defined in Williams et al. Streptomyces lavendulae is also classified in Cluster group F.
All known microbial transglutaminases have been identified by using a conventional enzyme assay in which hydroxylamine is converted to hydroxamic acid (Folk, J. E. & Cole, P. W. (1966)).
In order to construct strains overproducing different enzymes, recombinant DNA techniques are widely used. For the same purpose, the Streptoverticillium mobaraense transglutaminase gene has been cloned for expression in Escherichia coli, Streptomyces lividans, and Saccharomyces cerevisiae (Washizu et al., Tahekana et al., and EP-A-0 481 504). Even the most succesful of these approaches (Washizu et al.) resulted in a production yield much lower than the yield in the wildtype S. mobaraense strain, in spite of extensive experimentation and optimization. Thus, none of the efforts to overproduce the S. mobaraense enzyme have been successful, although they included a number of different approaches such as chemical synthesis of a codon-optimized gene and its subsequent expression (as a cleavable heterologous signal peptide fusion to the mature transglutaminase) to the periplasm of E. coli; or expression as a similar fusion to the mature transglutaminase in S. cerevisiae; or expression as a similar fusion to pro-transglutaminase in S. cerevisiae; or traditional isolation and expression of the natural DNA sequence encoding the preproenzyme in S. lividans.
U.S. Pat. No. 5,252,469 discloses strains closely related to S. mobaraense which produce higher amounts of transglutaminase by conventional techniques.
The object of the invention is to provide novel microbially derived transglutaminases, preferably in single-component or mono-component form, a novel gene encoding a transglutamin-ase, and a method for producing the transglutaminase in a better yield and higher purity than hitherto possible by recombinant DNA technology, as well as the use of the transglutaminase either alone or in combination with other enzymes for the use in a variety of industrial purposes, including gelling of proteins; improvement of baking quality of flour; producing paste type food or food ingredients from protein, fat and water; preparation of cheese from milk concentrate; binding of chopped meat or fish products; improvement of taste and texture of food proteins; casein finishing in leather processing; shoe shine, etc.